Na+/K+-ATPase with a Blocked E1ATP Site Still Allows Backdoor Phosphorylation of the E2ATP site

Abstract

The role of simultaneously existing ATP-binding sites in the catalytic process of Na+/K(+)-ATPase is unclear. In order to learn whether blocking the E1ATP site affects the properties of the E2ATP site, the E1ATP site was inactivated by either fluorescein 5'-isothiocyanate, the non-phosphorylating Cr(H2O)4AdoPP[CH2]P or the phosphorylating Cr(H2O)4ATP. The properties of the remaining E2ATP site were studied by measuring 'backdoor phosphorylation' in the presence of ouabain, or K(+)-activated hydrolysis of p-nitrophenyl phosphate. The involvement of the E2ATP site was further tested by the effects of Co(NH3)4ATP, a specific inactivator of this site. When the E1ATP site was inactivated by fluorescein 5'-isothiocyanate or the non-phosphorylating Cr(H2O)4AdoPP[CH2]P, backdoor phosphorylation and the activity of K(+)-activated p-nitrophenylphosphatase remained unchanged. Both processes were lost, however, when the E2ATP site was additionally inactivated by Co(NH3)4ATP. Inactivation of the E1ATP site by fluorescein 5'-isothiocyanate or Cr(H2O)4AdoPP[CH2]P decreased the affinity of the p-nitrophenylphosphatase activity of the E2ATP site for the substrate p-nitrophenyl phosphate by four times. This is consistent with a former report showing that dephosphorylation in a fluorescein 5'-isothiocyanate-inactivated Na+/K(+)-ATPase has a lowered sensitivity for ATP [Scheiner-Bobis, G., Antonipillai, J. & Farley, R. A. (1993) Biochemistry 32, 9592-9599]. Inactivation of the E1ATP site by the phosphorylating Cr(H2O)4ATP, however, led to a loss of the property of the E2ATP site to hydrolyse K(+)-dependent p-nitrophenyl phosphate and to achieve backdoor phosphorylation. Evidently, ATP sites coexist in Na+/K(+)-ATPase, and binding of ATP to one site affects the property of the other site [Scheiner-Bobis, G., Esmann, M. & Schoner, W. (1989) Eur. J. Biochem. 183, 173-178]. Although the enzyme can be phosphorylated from both ATP sites, phosphorylation of the E1ATP site excludes the phosphorylation of the E2ATP site.

Quenching of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐modified Na+/K+‐ATPase reveals a higher accessibility of the low‐affinity ATP‐binding site

“…1, insert) and implied a two-site modification process. T o learn whether NBD-Cl may affect the partial activity of the E2ATP site we studied the inactivation of K + -activated para-nitrophenylphosphatase by this reagent (Table 1) in a FITC-prelabeled N a + / K + -A T P a s e where the E j A T P site was thus blocked [2]. All inactivation effects of NBD-Cl on the partial activities could be prevented by millimolar concentrations of ATP (data not shown).…”

“…Both the spectral shift and the appearance of fluorescence, were a clear indication of NBD-Cl modifications of the enzyme. This was observed when the enzyme was labeled either with or without Co(NH 3 ) 4 ATP or Cr(H 2 0) 4 Ado/ , P[CH 2 ]P. The former compound is known as a specific inhibitor of the E 2 ATP site [2,5,7] while the latter blocks specifically the EiATP site [2,3]. The absorption or fluorescence spectra of enzyme-bound NBD-Cl did not differ significantly if the labeling of the enzyme was done in the presence of Co(NH3) 4 ATP or Cr(H 2 0) 4 AdoPP[CH 2 ]P (EjATP site or E 2 ATP site is blocked).…”

“…After incubation for 1 h at 37°C the enzyme was spun down, washed twice with 20 mM Tris/HCl and K + -activated para-nitrophenylphosphatase was estimated. For details see [2].…”

“…The process of cation transport has been described by the so-called Albers-Post model in which a single ATP site per catalytic a subunit exists in two main conformations which differ in their affinities for ATP: The high-affinity EiATP site is associated with Na + export while the low-affinity E2ATP site is implicated in K + import [1]. This model, however, is inconsistent with the observations of backdoor phosphorylation and of [ 86 Rb] occlusion in an EiATP site blocked enzyme [2,3] and with the 1 This work is part of the Ph.D. thesis of H.L.…”

“…Abbreviations: Cr(H20)4Adoi , i > [CH 2 ]i', ß,y bidentate complex of chromium(III)-tetraaqua-adenylyl [ß,y-methylene] diphosphonate; CofNHs^ATP, ß,y bidentate complex of cobalt(III)-tetramino-adenosine-5'-triphosphate; Co(NH3)4P04, tetramine cobalt(III)phosphate; FITC, fluorescein 5'isothiocyanate; NBD-C1, 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole, 7-chloro-4-nitro-benzofurazane; EiATP site, nucleotide-binding site of Na + /K + -ATPase with high affinity for ATP; E2ATP site, nucleotide-binding site of Na + /K + -ATPase with low affinity for ATP phenomena of superphosphorylation from [y-32 P]ATP [4], double labeling with ATP analogs [5] or double phosphorylation from para-nitrophenylphosphate [6]. All these observations, including the observation of a positive cooperative effect of 2'-ODANS-8-N 3 -ATP in the inactivation of Na+/K+-ATPase [7] led to the conclusion that two simultaneously existing ATP sites need to cooperate during ATP-driven Na+/ K + transport [7,8].…”